Johannes M. Weiss

An unrestrained proinflammatory M1 macrophage population induced by iron impairs wound healing in humans and mice

Uncontrolled macrophage activation is now considered to be a critical event in the pathogenesis of chronic inflammatory diseases such as atherosclerosis, multiple sclerosis, and chronic venous leg ulcers. However, it is still unclear which environmental cues induce persistent activation of macrophages in vivo and how macrophage-derived effector molecules maintain chronic inflammation and affect resident fibroblasts essential for tissue homeostasis and repair. We used a complementary approach studying human subjects with chronic venous leg ulcers, a model disease for macrophage-driven chronic inflammation, while establishing a mouse model closely reflecting its pathogenesis. Here, we have shown that iron overloading of macrophages--as was found to occur in human chronic venous leg ulcers and the mouse model--induced a macrophage population in situ with an unrestrained proinflammatory M1 activation state. Via enhanced TNF-α and hydroxyl radical release, this macrophage population perpetuated inflammation and induced a p16(INK4a)-dependent senescence program in resident fibroblasts, eventually leading to impaired wound healing. This study provides insight into the role of what we believe to be a previously undescribed iron-induced macrophage population in vivo. Targeting this population may hold promise for the development of novel therapies for chronic inflammatory diseases such as chronic venous leg ulcers.

Osteopontin and the skin: multiple emerging roles in cutaneous biology and pathology

Abstract:  Osteopontin (OPN) is a glycoprotein expressed by various tissues and cells. The existence of variant forms of OPN as a secreted (sOPN) and intracellular (iOPN) protein and its modification through post‐translational modification and proteolytic cleavage explain its broad range of functions. There is increasing knowledge which receptors OPN isoforms can bind to and which signaling pathways are activated to mediate different OPN functions. sOPN interacts with integrins and CD44, mediates cell adhesion, migration and tumor invasion, and has T helper 1 (Th1) cytokine functions and anti‐apoptotic effects. iOPN has been described to regulate macrophage migration and interferon‐α secretion in plasmacytoid dendritic cells. Both sOPN and iOPN, through complex functions for different dendritic cell subsets, participate in the regulation of Th cell lineages, among them Th17 cells. For skin disease, OPN from immune cells and tumor cells is of pathophysiological relevance. OPN is secreted in autoimmune diseases such as lupus erythematosus, and influences inflammation of immediate and delayed type allergies and granuloma formation. We describe that OPN is overexpressed in psoriasis and propose a model to study OPN function in psoriatic inflammation. Through cytokine functions, OPN supports immune responses against Mycobacteria and viruses such as herpes simplex virus. OPN is also implicated in skin tumor progression. Overexpression of OPN influences invasion and metastasis of melanoma and squamous cell carcinoma cells, and OPN expression in melanoma is a possible prognostic marker. As OPN protein preparations and anti‐OPN antibodies may be available in the near future, in‐depth knowledge of OPN functions may open new therapeutic approaches for skin diseases.

Keratinocytes Determine Th1 Immunity during Early Experimental Leishmaniasis

Experimental leishmaniasis is an excellent model system for analyzing Th1/Th2 differentiation. Resistance to Leishmania (L.) major depends on the development of a L. major specific Th1 response, while Th2 differentiation results in susceptibility. There is growing evidence that the microenvironment of the early affected tissue delivers the initial triggers for Th-cell differentiation. To analyze this we studied differential gene expression in infected skin of resistant and susceptible mice 16h after parasite inoculation. Employing microarray technology, bioinformatics, laser-microdissection and in-situ-hybridization we found that the epidermis was the major source of immunomodulatory mediators. This epidermal gene induction was significantly stronger in resistant mice especially for several genes known to promote Th1 differentiation (IL-12, IL-1β, osteopontin, IL-4) and for IL-6. Expression of these cytokines was temporally restricted to the crucial time of Th1/2 differentiation. Moreover, we revealed a stronger epidermal up-regulation of IL-6 in the epidermis of resistant mice. Accordingly, early local neutralization of IL-4 in resistant mice resulted in a Th2 switch and mice with a selective IL-6 deficiency in non-hematopoietic cells showed a Th2 switch and dramatic deterioration of disease. Thus, our data indicate for the first time that epidermal cytokine expression is a decisive factor in the generation of protective Th1 immunity and contributes to the outcome of infection with this important human pathogen.

Reactive oxygen intermediate-induced pathomechanisms contribute to immunosenescence, chronic inflammation and autoimmunity

Deregulation of reactive oxygen intermediates (ROI) resulting in either too high or too low concentrations are commonly recognized to be at least in part responsible for many changes associated with aging. This article reviews ROI-dependent mechanisms critically contributing to the decline of immune function during physiologic - or premature - aging. While ROI serve important effector functions in cellular metabolism, signalling and host defence, their fine-tuned generation declines over time, and ROI-mediated damage to several cellular components and/or signalling deviations become increasingly prevalent. Although distinct ROI-associated pathomechanisms contribute to immunosenescence of the innate and adaptive immune system, mutual amplification of dysfunctions may often result in hyporesponsiveness and immunodeficiency, or in chronic inflammation with hyperresponsiveness/deregulation, or both. In this context, we point out how imbalanced ROI contribute ambiguously to driving immunosenescence, chronic inflammation and autoimmunity. Although ROI may offer a distinct potential for therapeutic targeting along with the charming opportunity to rescue from deleterious processes of aging and chronic inflammatory diseases, such modifications, owing to the complexity of metabolic interactions, may carry a marked risk of unforeseen side effects.

Effect of Osteopontin Alleles on β-Glucan-Induced Granuloma Formation in the Mouse Liver

The granuloma formation is a host defense response against persistent irritants. Osteopontin is centrally involved in the formation of granulomas. Three osteopontin alleles, designated a, b, and c, have been found in mice. Here we used a murine model of zymosan (beta-glucan)-induced granuloma formation in the liver to determine possible functional differences between the osteopontin alleles in cell-mediated immunity. In contrast to mice with alleles a or c, mice with the allele b was defective in granuloma formation. As detected by mRNA expression, cytokines and chemokines known to be critically involved in granuloma formation were elicited in liver tissue, regardless of the osteopontin allele expressed. Alignment of the deduced amino acid sequences showed that unlike osteopontin c, b differs from a in 11 amino acids. All three osteopontin alleles had normal cell-binding properties. However, only the b allelic form was defective in the induction of cell migration as tested with dendritic cells. In conclusion, generation of a granulomatous response in mice depends critically on the presence of a functional osteopontin allele. Defective granuloma formation in mice with allele b is likely to be because of an impaired chemotactic function of the osteopontin b protein on immunocompetent cells.

Post‐marketing surveillance on treatment of 5,665 patients with atopic dermatitis using the calcineurin inhibitor pimecrolimus: Positive effects on major symptoms of atopic dermatitis and on quality of life

Background: Topical application of the calcineurin inhibitors pimecrolimus and tacrolimus is a current major advance in the therapy of atopic dermatitis. The aims of this post‐marketing surveillance were: a) to acquire data on the efficacy and tolerability of pimecrolimus ointment (Elidel®) on a very large cohort of patients from outpatient clinics, and b) to assess changes in their quality of life, a parameter not often considered in previous studies.

Osteopontin attenuates aging‐associated phenotypes of hematopoietic stem cells

Upon aging, hematopoietic stem cells (HSCs) undergo changes in function and structure, including skewing to myeloid lineages, lower reconstitution potential and loss of protein polarity. While stem cell intrinsic mechanisms are known to contribute to HSC aging, little is known on whether age‐related changes in the bone marrow niche regulate HSC aging. Upon aging, the expression of osteopontin (OPN) in the murine bone marrow stroma is reduced. Exposure of young HSCs to an OPN knockout niche results in a decrease in engraftment, an increase in long‐term HSC frequency and loss of stem cell polarity. Exposure of aged HSCs to thrombin‐cleaved OPN attenuates aging of old HSCs, resulting in increased engraftment, decreased HSC frequency, increased stem cell polarity and a restored balance of lymphoid and myeloid cells in peripheral blood. Thus, our data suggest a critical role for reduced stroma‐derived OPN for HSC aging and identify thrombin‐cleaved OPN as a novel niche informed therapeutic approach for ameliorating HSC phenotypes associated with aging.

Osteopontin and allergic disease: pathophysiology and implications for diagnostics and therapy

Osteopontin (OPN) is a phosphoglycoprotein that is expressed by various immune cells in a secreted and intracellular form. It has cytokine, chemotactic and cell signaling functions enhancing Th1 and Th17 immunity and protects against apoptosis. Recent studies found OPN to be modulatory in cell-mediated and immediate-type allergic diseases. In allergic asthma, OPN enhances sensitization but downmodulates Th2-driven IL-4-dominated inflammation. The finding that OPN expression is augmented during specific immunotherapy supports a Th2 suppressive effect of OPN. In Th1-driven delayed-type allergy, such as allergic contact dermatitis, OPN supports dendritic cell migration and IL-12 expression and is secreted by T effector cells and keratinocytes, augmenting Th1-mediated allergy and supporting disease chronification. There are numerous missing links as to how OPN variants modulate allergic inflammation through different OPN receptors. OPN research in allergy is an interesting, rapidly expanding field that has high potential for translational research.

Methylenetetrahydrofolate reductase polymorphism associated with moderate hyperhomocysteinaemia in a patient with livedo vasculopathy: treatment with vitamin supplementation and low molecular weight heparin

SIR, Livedo vasculopathy, a distinct form of thrombogenic vasculopathy, is characterized by recurrent painful ulcers of the lower extremity with persistent livedo racemosa. High levels of homocysteine are an independent risk factor for arterial occlusive diseases and deep vein thrombosis. One study demonstrated a possible association between plasma homocysteine levels and livedo vasculopathy. The key enzyme controlling remethylation of homocysteine is 5,10-methylenetetrahydrofolate reductase (MTHFR). An alanine/valine gene polymorphism caused through a C to T substitution at nucleotide 677 is responsible for a reduced MTHFR activity and increased thermolability of this enzyme in homozygous individuals, resulting in increased plasma homocysteine concentrations, particularly under conditions of low dietary folate intake. We report livedo vasculopathy in a patient with moderate hyperhomocysteinaemia due to homozygous MTHFR gene mutation. A 42-year-old woman presented with livedo racemosa and several small bizarrely shaped ulcers and atrophie blanche on the dorsal aspect of both feet and ankles (Fig. 1). Dorsal arteries of both feet were palpable. Colour-coded duplex sonography and Doppler sonography revealed no signs of venous insufficiency. Ulcers had previously occurred during the summer months and healed with ivory-coloured scars. The patient denied other medical problems, intake of medications, vitamin supplements, alcohol or drugs, but smoked five cigarettes per day. Skin biopsy showed segmental hyalinization of the vascular wall, endothelial proliferation and formation of fibrin thrombi within superficial and deep dermal vessels (Fig. 2). Laboratory analysis showed normal results for erythrocyte sedimentation rate, differential blood count, partial thromoboplastin time, thrombin time, international normalized ratio, serum creatinine, liver enzymes, C-reactive protein, anticardiolipin antibodies (IgG and IgM), b2-glycoprotein I, rheumatoid factor, C3, C4, immune complexes, protein S, protein C, antithrombin III, factor V Leiden, antinuclear antibodies, anti-DNA, antineutrophil cytoplasmic antibodies, cryoglobulin, cold agglutinins and lupus anticoagulants. However, plasma homocysteine levels were elevated to 17Æ1 lmol L (normal 5Æ0–12Æ0). Vitamin B12 (251 pmol L ; normal 148–738) and folate (8Æ2 nmol L; normal 7–36) were within normal range but were close to the lower limit. Subsequently, polymerase chain reaction analysis from peripheral blood revealed a homozygous mutation in codon 677 of the MTHFR gene. Various pathogenetic causes associated with defective coagulation have been proposed for livedo vasculopathy, such as antiphospholipid antibodies, defective release of tissue plasminogen activator, protein C or S deficiency, abnormal platelet function and factor V Leiden mutation. In our patient we found mild homocysteinaemia and a homozygous MTHFR polymorphism C677T. MTHFR catalyses the FADH-linked reduction of 5,10methylenetetrahydrofolate to 5-methyltetrahydrofolate, the predominant circulatory form of folate and carbon donor for the remethylation of homocysteine to methionine. Individuals Fig 1. Painful ulcerations, atrophie blanche and livedo reticularis on the dorsal foot (a) and medial malleolus (b). Detail of the ulcer on the dorsal foot (c).

Regulated Osteopontin Expression by Dendritic Cells Decisively Affects their Migratory Capacity

TO THE EDITOR Osteopontin is a secreted arginine-glycine-aspartate (RGD)-containing phosphoglycoprotein that interacts with different cell-surface receptors, including αvβ3, αvβ5, α4β1, α9β1, and certain CD44 isoforms (Denhardt et al., 2001; Sodek et al., 2006; Scatena et al., 2007). Osteopontin (OPN) has been implicated to have important cytokine and chemokine functions in Th1/Tc1-mediated immunity against viral and bacterial pathogens and in type-1-mediated diseases such as rheumatoid arthritis, autoimmune encephalitis, and granuloma formation (Ashkar et al., 2000; Diao et al., 2004; Tanaka et al., 2004; Shinohara et al., 2005; Hur et al., 2007). We demonstrated that OPN-null mice are impaired in their capacity to mount allergic contact hypersensitivity against trinitro-chlorobenzene (TNCB) (Weiss et al., 2001). The initiation of Th1-mediated immunity depends upon the function of antigen-presenting myeoloid dendritic cells (DCs) (Banchereau et al., 2000; Kapsenberg 2003). Contact hypersensitivity is a Th1-mediated immune response that is elicited once a hapten has been recognized during the initial sensitization phase. DCs are central to the sensitization process, as they recognize and transport the allergen into skin draining lymph nodes for presentation to naive T cells (Romani et al., 2006). OPN is likely to influence contact hypersensitivity response because it activates DCs and induces their maturation toward an IL-12 secreting, Th1-polarizing phenotype (Renkl et al., 2005). We previously described that OPN is chemotactic for DCs and OPN-null mice are impaired in their capacity to attract DCs to skin draining lymph nodes (Weiss et al., 2001). As we and others found that myeloid DCs produce OPN when differentiating from monocytes, we went on to investigate the role of autocrine effects of OPN produced by DCs themselves (Kawamura et al., 2005; Renkl et al., 2005). As convincing evidence exists that the migratory phenotype of macrophages, endothelial cells, and various types of tumour cells is associated with a high OPN-expressing phenotype, we determined which factors regulate OPN expression during murine DC maturation and investigated the role of DC-expressed OPN for their migratory abilities (Bruemmer et al., 2003; Rangaswami et al., 2006; Sodek et al., 2006; Nystrom et al., 2007). In murine bone marrow cultures, we found that under the addition of GM-CSF and IL-4 OPN mRNA and protein secretion increased constantly from day 3 to 7 of DC maturation (Figure 1a and b). When separating CD11c+DC from bone marrow cultures, these were the major OPN-secreting cells as they expressed high levels of OPN mRNA in contrast to the CD11c-depleted fraction (Figure 1c). Our findings demonstrate that OPN expression is strongly upregulated early during DC differentiation from bone marrow precursors. The fact that immature DCs highly express OPN is interesting especially as other immune cells, such as T cells, NK-cells, and monocytes, express OPN only following their activation in an inflammatory context (Shinohara et al., 2005; Sodek et al., 2006; Hur et al., 2007). Figure 1 Upregulation of constitutive OPN secretion by TNF-α and IL-1α correlates with the migratory capacity of DCs Both tumor-necrosis factor-α (TNF-α) and IL-1α induce a highly migratory phenotype in DCs (Cumberbatch et al., 2002). To establish a possible correlation between the migratory phenotype of DCs and their OPN-secreting potential, DCs were stimulated with TNF-α, IL-1α, or lipopolysaccharide (LPS). TNF-α and IL1-α induced OPN secretion (Figure 1d and e). Whereas IL-4 only moderately downmodulated OPN expression, LPS that induces terminally activated, non-migratory DCs strongly inhibited their OPN secretion, which was not due to significantly reduced viability (Figure 1d and e). In Boyden chamber assays, we investigated how the level of OPN secretion correlates with the migratory potential of DCs (Figure 1e). Indeed, the highly OPN-secreting, TNF-α- or IL-1α-treated DCs efficiently migrated toward TNF-α (Figure 1e). In contrast, LPS-matured, low OPN secreting DCs were non-migratory (Figure 1e). Similar to our findings with DCs, it was described that LPS suppresses OPN in macrophages (Shinohara et al., 2006), however, not correlating this OPN-low state with migration. We can only speculate that LPS in both cell types may induce a low OPN-expressing less migratory phenotype. To demonstrate that OPN deficiency is sufficient to influence DC migratory function, DCs generated from wild type and OPN-null mice were compared in their potential to migrate toward TNF-α, OPN, and CCL19. Whereas OPN wild-type DCs showed the previously described migration toward TNF-α, OPN, and CCL19, OPN-deficient DCs were unable to sufficiently migrate toward any of these stimuli (Figure 2a and b). Interestingly, in both monocytes and DCs, the high OPN-expressing state is correlated with high mobility. In vivo, macrophages migrating toward dermally injected N-formyl-met-leu-phe highly express OPN and Nystrom et al. (2007) recently reported that silencing of OPN expression by RNA interference impaired macrophage migration in vitro (Giachelli et al., 1998). Figure 2 OPN-deficient DCs are impaired in their capacity to migrate in vitro and to enter skin draining lymph nodes in vivo To demonstrate that OPN expression also correlates with DC migratory potential in vivo, fluorescently labeled wild-type DCs or OPN-deficient DCs were injected into the abdominal skin of wild-type mice (Lappin et al., 1999). Quantitative fluorescence-activated cell sorting analysis of pooled inguinal and axillary lymph nodes revealed that OPN-deficient DCs are significantly limited in their migratory efficiency into skin draining lymph nodes (Figure 2c). These data provide evidence that autocrine OPN production is centrally involved in DC migratory abilities. The mechanism by which OPN mediates DC migration remains to be elucidated. Chemokine receptors CCR5 and CCR7 are both crucially involved in migration of DCs. We speculated that these receptors are modulated by OPN. However, DC treatment by recombinant OPN revealed that their expression is not influenced by OPN (data not shown). In addition to the secreted form, an intracellular form of OPN may be important for DC migration by modulating CD44 activity, as described for peritoneal macrophages (Zohar et al., 2000; Zhu et al., 2004). Because we have previously shown that CD44 isoforms are involved in DC migration, further investigations on the role of the intracellular form of OPN in DC migration are under way (Weiss et al., 1997). In conclusion, our work demonstrates that OPN is highly expressed by myeloid DCs and is differentially modulated by DC-stimulating factors. In vivo and in vitro optimal DC migration depends on the expression of OPN.